The important biomedical scientific problem addressed by this proposal is the need for new drugs to treat metabolic and degenerative disorders related to oxidative stress. A spectrum of such multiple chronic disorders is associated with oxidative damage to many tissues and organs that are constantly challenged by environmental and endogenous stressors. Cells protect themselves from oxidative damage by activation of antioxidant genes in response to oxidative stress, but during the aging process as well as under certain situations where stress is excessive, the protective response is impaired or inadequate. However, there is a fundamental lack of therapeutic agents against such damage. Therefore, drugs that selectively inhibit oxidative damage to specific tissues and organs would be of great therapeutic value. The physiological antioxidant response essentially involves transcriptional activation of a battery of genes driven by an antioxidant response regulatory element (ARE) through binding of the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2/ARE regulates expression of most antioxidant enzymes including the Pi class glutathione S-transferases (GSTP1s); indeed, gstp1 is strongly induced upon Nrf2 activation in vertebrates when the animals are treated with electrophiles. This proposal describes the development of a combinatorial sequential approach by using both cell-based and animal-based high-throughput screens that are uniquely feasible at Scripps Florida. In Aim 1, in addition to the traditional cel-based ARE-responsive reporter gene approach, a new cell-based Nrf2 degron- fused reporter strategy will be employed in order to identify drug leads that promote an endogenous response to oxidative stress and thereby prevent oxidative damage. These cell-based assays can be efficiently pursued by complimenting ultra-high throughput screening of a chemical library comprised of approximately 1 million compounds with structures known to have properties suitable for drug development. In Aim 2, a high-volume whole-organism screen, which we would have developed, using a new transgenic zebrafish expressing gstp1 promoter-driven enhanced green fluorescence protein (GFP) reporter system will be used in a live organism- oriented secondary screening strategy for pre-selected chemical compounds in Aim 1. This allows us to validate developmental toxicity and bioavailability of drug candidates to be addressed simultaneously. Validated chemical hits in the zebrafish embryo and larval screen can be further evaluated to determine organ specificity as the fish grow and for efficacy in preventing oxidative damage. Aim 3 is designed to understand how these selected small molecules work in vivo so that additional drugs can be designed based on the mechanism of activation of the antioxidant response. Chemical genetic approaches, such as selective chemical inhibitors against plausible signaling pathways as well as morpholino-based genetic manipulations in zebrafish will be exploited to establish which components of the antioxidant signaling pathway are modified by activators and/or potentiators identified in the screening assays. PUBLIC HEALTH RELEVANCE: There is an urgent unmet need to develop novel drugs for the treatment and/or prevention of metabolic and degenerative disorders and to elucidate the underlying causes of these disorders. In such disorders, loss of optimal functioning is associated with increased oxidative damage to cells and impairment of the cellular mechanisms and homeostasis that normally protect cells from oxidative stress. The objectives of this proposal are to 1) exploit new cell-based high- throughput screening assays to identify drug leads that enhance resistance to oxidative damage; 2) evaluate the efficacy of such compounds in physiologically relevant novel animals models; and 3) understand how these compounds act, to further develop and characterize a novel series of drugs for treating metabolic and degenerative disorders.